Validation of a qualitative real-time PCR assay for the detection of Candida auris in hospital inpatient screening

ABSTRACT Candida auris is a multidrug-resistant opportunistic fungal pathogen capable of causing serious infections and healthcare-associated outbreaks. Screening for colonization with C. auris has become routine and is recommended in many hospitals and healthcare facilities as an infection control and prevention strategy. Subsequently, and since there are currently no FDA-approved tests for this purpose, clinical microbiology laboratories have become responsible for developing protocols to detect C. auris using axial and inguinal screening swabs. In a College of American Pathologists-accredited large academic healthcare center setting, we implemented a laboratory-developed nucleic-acid amplification test for the detection of C. auris DNA. Our test validation evaluated the performance of the DiaSorin C. auris primer set used in a real-time qualitative PCR assay on the LIAISON MDX thermocycler with the Simplexa Universal Disc. The assay was highly sensitive and specific, with a limit of detection of 1–2 CFU/reaction, with no observed cross-reactivity with other Candida spp., bacterial skin commensal organisms or commonly encountered viruses. When run in parallel with a culture-based detection method, the PCR assay was 100% sensitive and specific. The assay was precise, with low variability between replicates within and between runs. Lastly, pre-analytical factors, including swab storage time, temperature, and transport media, were assessed and found to have no significant effect on the detection of C. auris at variable concentrations. Taken together, this study expands the available options for nucleic acid detection of C. auris and characterizes pre-analytical factors for implementation in both high- and low-volume laboratory settings. IMPORTANCE This study overviews the validation and implementation of a molecular screening tool for the detection of Candida auris in a College of American Pathologist-accredited clinical laboratory. This molecular laboratory-developed test is both highly sensitive and specific and has significant health-system cost-savings associated with significantly reduced turn-around-time compared to traditional standard-of-care culture-based work up. This method and workflow is of interest to support clinical microbiology diagnostics and to help aid in hospital inpatient, and infection prevention control screening.

spp., include the ability of C. auris to colonize skin, form persistent, disinfectant-resistant biofilms on healthcare surfaces, and rapidly develop resistance to multiple classes of antifungal agents.Together, these characteristics advantageously contribute to the organism's ability to inhabit and spread in both healthcare and LTC facilities.
Skin colonization represents the largest outbreak risk to clinical environments as asymptomatic carriers of C. auris can contaminate high-touch surfaces and be transfer red to other susceptible non-carriers (3).Additionally, in environments where C. auris enters a biofilm state, these pathogens resist common disinfectants, including chlorhexi dine, sodium hypochlorite, peracetic acid, and caspofungin (4,5).Given these factors, implementation of initiatives that aid identifying patient reservoirs of C. auris and engaging the appropriate infection prevention methods to control additional disper sal of this pathogen have become necessary.To combat the spread of C. auris in healthcare and LTC facilities, screening patients for C. auris colonization has become routine.Culture-based screening of patient axilla and inguinal swabs involves manual interpretation of media which select for and differentiate Candida spp.after 18-36 h of incubation (6)(7)(8).While reliable, this form of screening can create a large burden for clinical laboratories when screening all inpatient admissions.
Nucleic acid testing for C. auris offers a more directed approach with faster turnaround for patient screening.While there is no FDA-approved molecular assay for C. auris screening currently available, a validated form of TaqMan real-time PCR (rt-PCR) has been developed by the Wadsworth Center in New York that utilizes the BD MAX platform to analyze extracted nucleic acids from swab specimens (9,10).Other commercially available nucleic acid tests, including the OLM Diagnostics AurisID, BioGX Candida auris, and Fungiplex Candida auris, have been developed and are indicated for research use only within the United States.These tests use variously sourced materials for the detection of C. auris and have variable specificities and limits of detection (11).
In this work, we validated a C. auris nucleic acid test using the DiaSorin C. auris primer set on the DiaSorin LIAISON MDX and DiaSorin Simplexa Universal Disc plat form to create a direct-from-swab screening approach.Additionally, to demonstrate the robustness of this screening assay in operational scaling from daily runs to weekly pooled testing, we assessed the stability of mocked biological swabs stored at both ambient and refrigerated temperatures over the course of one week.Taken together, this work demonstrates a culture-free, highly sensitive and specific C. auris screening approach that can be integrated into both low-and high-volume laboratory operations while meeting minimal requirements for both the Clinical Laboratory Improvement Amendments (CLIA) and the College of American Pathologists (CAP).

Patient samples and controls
Clinical specimens were collected using BBL CultureSwabs in liquid Stuart or liquid Amies medium (Becton Dickinson, Franklin Lakes, NJ) or Transystem swabs in liquid Stuart medium (Copan, Murrieta, CA).Clinical specimens were collected for C. auris coloniza tion screening by swabbing a combination of axial, inguinal, and/or other external body sites.Clinical swab samples were inoculated to Sabouraud dextrose agar with chloramphenicol (Remel, San Diego, CA) for downstream identification via MALDI-TOF and Candida CHROMagar (Hardy Diagnostics, Springboro, OH) plates for culture-based C. auris screening according to the method developed by the U.S. Centers for Disease Control and Prevention (12).The same swabs were then processed for detection by PCR via the method described below.A total of 282 patient samples were enrolled for this study.C. auris positive controls were made from a stock of C. auris (MYA-5002; ATCC, Manassas, VA).This stock culture was quantified by viable plate count, diluted to a working concentration of 5 × 10 5 CFU/mL, and frozen at −80°C.All samples were prepared and manipulated within a biological safety cabinet in a BSL-2 laboratory with enhanced precautions.

Specimen preparation and heat lysis
Swabs were broken off into a 5 mL tube containing 500 µL of Tris-EDTA (TE) pH 8.0 and vortexed.Five microliters of Fungal Lysis Solution (DiaSorin, Cypress, CA) and 50 µL of the sample-containing TE buffer were added to a 2-mL screwcap tube and vortexed.This mixture was then incubated at 60 ± 2°C in a rocking incubator at 25 strokes/minute for 30 minutes.A C. auris positive control and no template control (NTC) were also prepared and lysed with the patient samples.If testing was to be performed within 24 hours of lysis, lysates were stored at 2-8°C.If testing was to be performed beyond 24 hours of lysis, lysates were stored at −80°C.

Qualitative real-time PCR
A PCR master mix was prepared using temperature-activated (TA) master mix (Dia Sorin, Cypress CA), C. auris primer pair (DiaSorin, Cypress, CA), Simplexa extraction and amplification control set (DiaSorin, Cypress, CA), and DNAse-free H 2 O.For each sample, 4 µL of TA Master Mix, 3.25 µL of DNAse free H 2 O, 0.40 µL of internal control DNA, 0.20 µL of C. auris Primer Pair, and 0.15 µL of internal control primer were combined to form the master mix.The master mix was briefly vortexed and centrifuged for homogeneity.Eight microliters of master mix and 2 µL of sample or control were aliquoted into each well of a Simplexa Universal Disc (DiaSorin, Cypress, CA).The Universal Disc was loaded on the LIAISON MDX thermocycler (DiaSorin, Cypress, CA) and run according to the following protocol: 10 minute initial activation step at 97°C, followed by 45 cycles of 10 second denaturation at 97°C and 30 seconds annealing at 60°C.Samples with a Ct value less than 40 were considered positive.

Sensitivity, specificity, and reproducibility
Analytical sensitivity was determined by creating a serial dilution of C. auris (MYA-5002; ATCC, Manassas, VA) from 10 7 to 10 2 CFU/mL as quantitated by viable plate count.The limit of detection (LOD) was determined by probit analysis with a 95% CI.Analytical precision was assessed by measuring the coefficient of variation (%CV) in triplicate of quantitated C. auris controls.Analytical specificity was determined by assessing microorganisms and viruses obtained from a combination of clinical specimens and reference samples (ATCC, Manassas VA; Zeptometrix, Franklin MA) at concentrations of 10 5 CFU/mL yeast and bacteria, 10 5 plaque forming units (PFU)/mL, TCID 50 /mL, or copies/mL for viruses.Yeast and bacteria were quantified by viable plate count and diluted to a final concentration using sterile saline.Viruses were obtained pre-titred and diluted to a final working concentration using sterile phosphate-buffered saline (PBS).Axial swabs from confirmed C. auris-negative volunteers were then inoculated with the non-specific analytes of interested and screened by the above-described methodology.
Clinical sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were determined by assessing 282 clinical specimens by both the real-time PCR assay and fungal culture as the gold standard comparator.Clinical specimens were plated on Sabouraud dextrose agar with chloramphenicol and HardyCHROM Candida agar plates.

Sample stability
To assess stability of the samples over time and in different transport media, mock high (10 7 CFU/mL) and low (10 4 CFU/mL) samples were created and stored in Liquid Stuarts, Liquid Amies, and Gel Amies transport media that contained axial swabs from people proven to be C. auris-negative.A subset of samples was tested immediately (Day 0), while the others were stored at 4°C or room temperature and tested on days 1, 2, 3, and 7.

Statistics
LOD was determined by probit analysis using MedCalc version 20.113 (MedCalc Software, Ostend, Belgium).For the sample stability analyses, one-way analysis of variance (ANOVA) was performed and visualized in Prism version 9.2.0 (Graphpad, Boston, MA).

Analytical sensitivity, specificity, and precision
Analytical sensitivity and precision were determined by assaying a dilution series ranging from 10 7 to 10 2 CFU/mL C. auris that was quantified by viable plate count.Analytical sensitivity of the assay, as determined by probit analysis is 879.5 CFU/mL with a 95% CI from 66.31 to 1497.51 CFU/mL (Fig. 1).This equates to 1-2 CFU/reaction, which is consistent with other published laboratory developed tests (LDTs) for C. auris screening from surveillance swabs via real-time PCR (9,10,13).The dilution series was performed in triplicate to determine precision (Table 1), which was measured by the coefficient of variance (%CV) of the generated C t values.For samples run on the same disk on the same day (intra-assay variation), %CV ranged from 0.56% to 4.94%.For samples run on different discs (inter-assay variation), %CV ranged from 0.60% to 7.90%.Analytical specificity was determined by performing the assay with no template controls (NTC), other Candida spp., skin commensal bacteria, and frequently encountered viruses.No amplification or cross-reactivity was observed with the NTC, microorganisms, or viruses tested, but the internal control was detected in each reaction (Table 2).

Sample stability
Sample stability over time, at different holding temperatures, and in different transport media were assessed to determine the effect these pre-analytical factors had on the assay results.No significant differences were observed between day 0 and day 7, at room temperature or at 4°C, across the different tested transport media (Fig. 2).At room temperature, Ct values ranged from 23.3 to 26.1 and 30.2 to 35.3 for the high-concentra tion and low-concentration sample swabs (Amies and Stuarts), respectively.At 4°C, Ct values ranged from 23.1 to 25.1 and 30.9 to 35.3 for the high concentration and low concentration sample swabs (Amies and Stuarts), respectively.

Clinical sensitivity and specificity relative to culture
Clinical sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were determined by assessing 282 clinical specimens by both the qualitative real-time PCR assay and fungal culture as the gold standard comparator.C. auris was detected by both conventional culture and rt-PCR assay in 23 total clinical samples, for a 100% clinical sensitivity rate.C. auris was not detected by culture or rt-PCR in 259 samples, for a 100% clinical specificity rate.Positive predictive value and NPV were also 100% (Table 3).

DISCUSSION
Since the emergence of C. auris, and subsequent implementation of infection control and prevention measures, screening of patients for C. auris colonization has created a new demand for clinical microbiology laboratories.While culture-based methods of detection are acceptable, the turn-around-time (TAT) from sample collection to result is 1-5 days, potentially delaying patient transfer to another unit or facility, or use of inappropriate contact precautions.Molecular assays are well-suited for screening tests such as this, as they can be scaled to handle large volumes, provide accurate results with a decreased TAT, and reduce overall healthcare spending.Although molecular tests are generally more expensive than culture-based tests, the decrease in TAT and subsequent reduction in hospital stay when a negative C. auris screen is required prior to discharge to a LTC facilities justify the more expensive test for an overall cost savings.
Without available FDA-approved molecular assays for C. auris detection, clinical laboratories must perform studies on LDTs to assess their performance if they wish to bring on testing.Minimum criteria that must be assessed in such a validation include analytical sensitivity and specificity, precision, LOD, and comparison to a gold stand ard method, as defined by both CAP and CLIA.In this validation study, these parame ters were consistent with, or improved, compared to other similar studies.Previously published validations have shown sensitivity ranging from 89% to 100% and specificity ranging from 92% to 100% (9,10,13,14).This assay demonstrated equivalent, or superior, sensitivity and specificity with both metrics at 100%.It is worth noting that the inter-assay %CV was determined to be 7.9% at a concentration of 10 7 CFU/mL, which can be concerning, particularly at higher concentrations.This was due to a single replicate (n = 15 total replicates per dilution) resulting in a Ct value of 19.5, which may be attributed to the inherent small volume handling of the DiaSorin assay.When this value is omitted, the adjusted calculated %CV was determined to be 2.7%.No cross-reactivity was observed with other Candida spp., including C. haemulonii and C. doubushaemulonii, which are closely related to C. auris and have had misidentifications documented (15,16).Additionally, no cross-reactivity was observed with bacterial skin commensal organisms, or with viruses and bacteria commonly encountered in clinical specimens, indicating the primers are highly specific for C. auris.
After evaluating the performance of the assay, we assessed sample stability over time, at two different temperatures, in different transport media, and at high and low analyte concentrations.These pre-analytical factors are important to evaluate for this test to withstand unexpected delays in testing and to allow for batching of samples.No changes in Ct values were observed between day 0 and day 7 in any of the condi tions tested, indicating that samples can be stored for at least a week before testing, if necessary.Furthermore, samples can be stored at room temperature or 4°C without affecting detection.The sample stability was consistent in all three transport media, providing greater flexibility for sample collection.
Replacing traditional culture with nucleic acid testing for C. auris carrier screening reduces a large labor burden to clinical laboratories by automating batch testing of samples.The robustness and commonplace nature of nucleic acid testing enables laboratories in sizes ranging from small community hospitals up to centralized clinical pathology facilities to bring this test in-house.One limitation of this type of testing compared to culture-based methodology is the analytical endpoint only assesses the presence or absence of the yeast and does not yield an isolate that could be used if C. auris antifungal susceptibility testing or outbreak tracing via whole-genome sequencing is necessary.A limitation of this study is the small number (n = 23) of positive clinical samples that were collected over the study period.A larger number of positive samples could impact the predictive values associated with this assay and increase the probability of detecting errors resulting in false negative or false positive results.Furthermore, at the time of study, CFU counts of the PCR-positive specimens were not performed.
This study assessed the performance of a commercially available primer set and reagents implemented for use on a commercially available platform.Other clinical microbiology laboratories could purchase these reagents and instruments and replicate this process to validate this assay in their own laboratories.Furthermore, pre-analytical factors were tested and shown not to have a significant influence on sample stability and downstream assay performance, increasing the utility and robust nature of this assay.Ultimately, this work addresses a pressing need for rapid, high-throughput testing options for C. auris colonization screening.

FIG 2
FIG 2 Sample stability over time at room-temperature (A) and 4°C (B) in Amies and Stuart's transport media.

TABLE 1
Reproducibility and repeatability of the C. auris real-time PCR assay a C.

TABLE 2
Analytical specificity was determined by screening for C. auris in specimens containing other Candida spp., bacterial skin flora, and clinically relevant viruses

TABLE 3
Performance of the C. auris real-time PCR assay compared to culture as gold standard testing